Electrospun fiber mats and methods for the making thereof

ABSTRACT

Disclosed herein are methods of forming a fiber mat, involving forming an aqueous solution of at least one protein, at least one polysaccharide, and optionally a plasticizer, and electrospinning the aqueous solution onto a collector to form a mat.

BACKGROUND OF THE INVENTION

Disclosed herein are methods of forming a fiber mat, involving formingan aqueous solution of at least one protein, at least onepolysaccharide, and optionally a plasticizer, and electrospinning theaqueous solution onto a collector to form a mat.

Casein represents approximately 80% of the protein content in milk. Itis composed of alpha s₁, alpha s₂, beta- and kappa-casein in the ratiosof approximately 40:10:35:12 (Fox, P. F., The milk protein system, In:Developments in Dairy Chemistry—4, Functional Milk Proteins, P. F. Fox,ed., Elsevier Applied Science, New York, 1989) and exists in a colloidalcomplex bound together by Ca—P linkages and hydrophobic interactions.Kappa-casein stabilizes the exterior of the micelle, preventingprecipitation of the other caseins through hydrophilic interactions.Because casein is a phosphoprotein, it binds Ca in proportion to thenumber of P groups and may also bind other minerals such as Zn. Becauseof its unique open structure, the casein micelle structure as it existsin milk has been proposed as a nano encapsulant for targeted nutritionalor drug delivery (Livney, Y. D., et al., Nanoencapsulation ofhydrophobic nutraceutical substances within casein micelles, XIVthInternational Workshop on Bioencapsulation, Lausanne, CH. 07-4 pg. 1-4(2006)).

Casein has very low amounts of cysteine and no disulfide linkages,giving it a random coil structure (Gennadios, A., et al., Chapter 9,Edible Coatings and Films Based on Proteins In Edible Coatings and Filmsto Improve Food Quality, J. M. Krochta et al. eds., Technomic PublishingCo., Inc., Lancaster, Pa., 1994) with very little secondary or tertiarystructure. The random coil structure is responsible for the ability ofcasein to form films. The large number of proline residues allowsbending of the protein chains but prevents building of secondarystructures. Casein is very sensitive to pH which dictates its structurein solution, and ultimately its function. At low pH, casein is typicallyin the form of aggregates because the negative charges on the casein areneutralized upon lowering of pH to the isoelectric point of 4.6, withdecreased repulsion between the side chains (Chakraborty, A., and S.Basak, J. Photochem. Photobiol. B, 87: 191-199 (2007)). Furthermore, theCa—P linkages are dissolved, releasing the individual casein and themicellar structure is lost (Gennadios et al. 1994). Treating milk withrennet produces rennet casein which retains the micellar structure.Gelled products such as yogurt and some cheeses are manufactured underlow pH conditions. Acid casein may be dried and used in food products orin nonfood applications. With the addition of a base such as Na orCa(OH)₂ to acid casein, at pH in the range from approximately 7 to 9,the casein is solubilized and electrostatic interactions are favoredover other interactions such as hydrophobic interactions and hydrogenbonding. The caseinate formed does not have micellar structure. Theseproperties in addition to the random coil structure have been exploitedto form edible films and coatings from calcium caseinate (CaCAS), CO₂casein, and sodium caseinate (NaCAS) (Tomasula, P. M., Using dairyingredients to produce edible films and biodegradable packagingmaterials, In: Dairy-derived ingredients—Food and nutraceutical uses, M.Corredig, ed., Woodhead Publishing Ltd and CRC Press LLC, Boca Raton,Fla., 2009).

Casein-based edible films have usually been made using a casting processin order to determine their properties. They have excellent tensile andoxygen barrier properties, making them excellent candidates for use in awide variety of applications (Krochta, J. M., E. A. Baldwin, M.Nisperos-Carriedo, eds, 1994, Edible Coatings and Films to Improve FoodQuality, Technomic Publishing Co., Inc., Lancaster, Pa.; Tomasula,2009). Because of their food-grade status, edible casein films have beenproposed for use as part of food systems to prevent migration ofcomponents, add to appearance, and to add antimicrobials or nutrients(McHugh, T. H., and J. M. Krochta, Food Technol., January 1994, pp.97-103). Casein and caseinates have long been used in wet spinningprocesses for the manufacture of casein fibers for woolen and silk-likefabrics, although they were treated with formaldehyde to harden thefibers (Sutermeister, E., and F. L. Browne, Casein and Its IndustrialApplications, Reinhold Publishing Corporation, New York, 1939). Caseinfibers have also been proposed for obtaining artificial food proteinfibers by spinneret wet spinning (Suckov, V. V., et al., Die Nahrung.,24: 893-897 (1980)).

Recently, electrospinning, a technology for making nonwoven mats fromcontinuous fibers with thicknesses on the nano or microscale, has beenused for applications ranging from building tissue engineering scaffoldsto use as filter media (Greiner, A., and J. H. Wendorff, Angew. Chem.Int. Ed., 46: 5670-5703 (2007)). The fibers have a high surface area, onthe order of 1000× greater than their volume, yielding electrospunproducts with increased surface efficiency compared to cast films.Electrospinning involves applying a high voltage to a solutioncontaining the polymer. As a solution that is spinnable is dischargeddropwise through a nozzle, the electric field causes the drop to form ina cone shape which then forms a continuous jet. The jet becomes narrowerand forms an open coil as it approaches a counter electrode. The solventsimultaneously evaporates, precipitating the polymer on the counterelectrode. The drop is balanced at the nozzle by its surface tension andis ejected when the electric field is opposed by the solutionelectrostatic forces that become larger than the surface tension(Greiner and Wendorff, 2007). For successful creation of fibers,electrospinning requires solubility in the solvent, the electric fieldneeds to exceed that of the surface tension at the nozzle to form thecone, and entanglement of the molecular chains of the polymer, which isa function of the viscosity of the solution (Stijnman, A. C., et al.,Food Hydrocolloids, 25: 1393-1398 (2011)). Uneven jet formation orelectro spraying results in fibers that are interspersed with beads andother shapes. While electrospinning has been successfully applied tosynthetic polymers, it has more recently been applied to naturalpolymers.

There are several examples of electrospinning of natural (non food) andsynthetic polymers in non aqueous solvents, but there are relatively fewexamples of natural polymers electrospun from aqueous solutions, whichwould include polysaccharides and proteins for food applications.Stinjman et al. (2011) found that the minimum requirements forelectrospinning of polysaccharides, such as the conditions under which ajet and then fibers were formed, were shear-thinning behavior at shearrates less than 1000 s⁻¹ and overlap concentration, a measure of thechain to chain interactions and entanglement. Under these conditions,fibers were formed from dextran and pullulan (PUL). Electro spunproteins have required use of a process aid or carrier such aspoly(ethylene oxide) (PEO), which has been used to electrospin severalproteins, polysaccharides, and cellulose derivatives that cannot beelectrospun alone (Alborzi, S., et al., J. Food Sci, 75: C100-107(2010)). PEO is believed to lower the surface tension and electricalconductivity of the solution, thus enabling electrospinning of themixture. However, proteins electrospun with PEO are not edible.

Electro spinning of proteins without a carrier has been demonstrated forzein and gelatin. Zein was electrospun from 70% EtOH solutions (Miyoshi,T., et al., Polymer International., 54: 1187-1190 (2005);Kanjanapongkul, K., et al., J. Appl. Poly. Sci., 118: 1821-1829 (2010))and gelatin was electrospun from water only (Zhang, S., et al., J.Biomedical Materials Research, Part A, 90: 671-679 (2009). Gelatin at a12.5 wt % concentration (150A, 75B gelatin) was also used as a carrierfor electrospinning of proteins such as whey protein isolate, ovalbumin,BSA, soy protein isolate, and NaCAS, with optimal spinning temperatureof 40° C. (Nieuwland, M., et al., Innovative Food Sci. and EmergingTech., 20: 269-275 (2013)). The ability to electrospin a particularprotein was also found to be related to an ultrasonic treatment that wasrequired to disrupt aggregated proteins. A harsh treatment of theNaCAS-gelatin solution was required prior to electro spinning.

We have determined the molecular parameters and the operating conditionsnecessary to electrospin aqueous solutions of proteins (e.g., CaCAS,NaCAS) and polysaccharides (e.g., pullulan) for potential foodapplications The proteins may also be blended together and fats withmilk fats added to form emulsified structures.

SUMMARY OF THE INVENTION

Disclosed herein are methods of forming a fiber mat, involving formingan aqueous solution of at least one protein, at least onepolysaccharide, and optionally a plasticizer, and electrospinning theaqueous solution onto a collector to form a mat.

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the detaileddescription. This summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended asan aid in determining the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEM of electrospun fibers obtained from 5, 10 and 15% PULsolutions as described below: (A) 5% PUL solution (1000×), (B) 10% PULsolution (2500×), (C) 15% PUL solution (25,000×). Electrospinning wasconducted at 40° C. and at 3 mL/hr. Voltage was 10.5 kV for (A) and (C)and 8 kV for (B).

FIG. 2 shows SEM of electrospun fibers obtained from either 15% PUL or30% PUL solutions and 20% CaCAS solutions in various volume ratios asdescribed below: (A) 15% PUL; 20% CaCAS solutions (1:1)(5000×), (B) 15%PUL; 20% CaCAS solution (1:2) (5000×), (C) 30% PUL; 20% CaCAS (1:1)(2500×), (D) 30% PUL, 20% CaCAS solution (1:2)(5000×), and (E) 30% PUL,20% CaCAS (1:4)(5000×).

FIG. 3 shows SEM of electrospun fibers obtained from either 15% PUL and20% NaCAS solutions in various volume ratios #70-73 as described below:(A) (2:1) (10,000×), (B) (1:1) (25,000×), (C) (1:2)(5000×) (D) (1:4)(5000×).

FIG. 4 shows SEM (50,000×) of a dehydrated NaCAS fiber obtained from afiber that was electrospun from a 15% PUL and 25% NaCAS solution withvolume ratio (1:4) and then was immersed in EtOH/Glutaraldehyde (3%) for4 h, washed with EtOH and then H₂O as described below.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are methods of forming a fiber mat, involving formingan aqueous solution of at least one protein, at least onepolysaccharide, and optionally a plasticizer and electrospinning saidaqueous solution onto a collector (e.g., rotating cylinder or drum) toform a mat. At this stage we do not collect a single fiber on, forexample, a spool; we produce the fibers onto a defined area which may befixed or moving, such as on a rotating cylinder or drum. The fibers arelaid over the surface creating the mat.

The method generally utilizes a needle and a voltage source connected tothe needle, wherein the distance between the tip of the needle and thecollector is about 12 cm to about 15 cm (e.g., 12-15 cm). The voltagesource generally provides about 23 kV. The method can also involvesoaking the mat in a solution containing ethanol and glutaraldehyde toremove polysaccharides.

The pure aqueous solutions may contain up to about 20% by weight of theprotein, or up to about 50% of the polysaccharide. In solutionscontaining mixtures of the two, the solution may contain about 20%protein and about 20% of the polysaccharide. The solutions of either thepolysaccharides or proteins were used as our source solutions which wethen blended together to get our solution containing both the proteinand polysaccharide.

Proteins which may be used in the method include, for example, foodgrade proteins such as caseinates (e.g., calcium caseinate, CO₂ casein,sodium caseinate), acid casein, rennet casein, the individual caseinproteins such as alpha, beta and kappa-caseins; skim and milk thatcontains fats such as whole milk or low fat milk and milk proteinconcentrates; pre fermented or post-fermented milk containing dairycultures, probiotics, prebiotics, or fibers; milk containing wheyprotein concentrates, whey protein isolates, the individual wheyproteins such as beta-lactoglobulin, alpha-lactalbumin, BSA, soy proteinisolate, corn protein isolate, and the like; casein and whey peptidessolutions, alone or in combination; cheese milk; amino acids, or invarious mixtures of the above.

Polysaccharides which may be used in the method include, for example,food grade polysaccharides such as pullulan, dextran, pectin, chitosan,lactose, lactulose, and other biopolymers. The proteins may also beblended together and fats (e.g., milk fats) added to form emulsifiedstructures.

Plasticizer which may be used in the method include, for example,glycerol, water, sorbitol, polyethylene glycol, propylene glycol, sugarssuch as glucose and sucrose, monosaccharides, oligosaccharides, lipidssuch as monoglyceride, acetylated monoglycerides, and fatty acids suchas lauric acid, linoleic acid, stearic acid, and others known in theart.

The method does not utilize a process aid or carrier such aspoly(ethylene oxide) (PEO).

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances in which said event or circumstance occurs and instances whereit does not. For example, the phrase “optionally comprising a defoamingagent” means that the composition may or may not contain a defoamingagent and that this description includes compositions that contain anddo not contain a foaming agent.

By the term “effective amount” of a compound or property as providedherein is meant such amount as is capable of performing the function ofthe compound or property for which an effective amount is expressed. Aswill be pointed out below, the exact amount required will vary fromprocess to process, depending on recognized variables such as thecompounds employed and the processing conditions observed. Thus, it isnot possible to specify an exact “effective amount.” However, anappropriate effective amount may be determined by one of ordinary skillin the art using only routine experimentation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. As used herein, the term “about”refers to a quantity, level, value or amount that varies by as much as30%, preferably by as much as 20%, and more preferably by as much as 10%to a reference quantity, level, value or amount. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

The following examples are intended only to further illustrate theinvention and are not intended to limit the scope of the invention asdefined by the claims.

EXAMPLES

Materials and Methods: Sodium caseinate (NaCAS) and calcium caseinate(CaCAS) were obtained from the American Casein Co. (Burlington, N.J.).According to the manufacturer, NaCAS contained 93% protein (dry basis)or 89% protein (as is), fat 1.5%, ash 3.5%, carbohydrate<1%, andmoisture 5%; pH was 6.9. CaCAS contained 91% protein on a dry basis and88% as is, fat 1.7%, ash 4.0%, carbohydrate<1%, and moisture 5%; pH was6.9. Pullulan (PUL) was obtained from TCI America (Portland, Oreg.). Allmaterials were used without further modification or purification. Thedeionized water used in all solutions was produced using a BarnsteadE-pure water system (Dubuque, Iowa).

Stock solutions of aqueous CaCAS or NaCAS were prepared containingeither 15 or 20% (w/w). These were diluted with solutions of PULprepared at concentrations ranging from 3 to 15% (w/w). Solutions ofCaCAS/PUL or NaCAS/PUL were prepared with deionized water to totalsolids concentrations ranging from 1.7 to 20% (w/w). The concentrationof CaCAS or NaCAS was 20% and that of PUL was 15% in CAS/PUL solutions.After the addition of the caseinates, polysaccharides, and optionallyglycerol to water, the solutions were mechanically stirred (Cole-Parmer,Vernon Hills, Ill.) for 1 to 2 hrs at 1000 rpm. The solutions wererefrigerated overnight at 4° C. to remove air bubbles.

Fibrous Mat preparation: Electro spinning experiments were conductedusing a nanofiber electrospinning unit (NaBond Technologies, Hong Kong,China; http://www.nabond.com). This unit consists of a cabinet housingelectrospinning equipment which included the following:

a syringe pump leading to a needle (or the functional equivalent); acontrollable high voltage source connecting to the needle; and arotating collector to collect the fibers. The solution containing CASwas added to the syringe pump prior to the experiments. The collectorwas wrapped in aluminum foil to facilitate collection of the fibrousmats produced.

Preliminary experiments were conducted by varying the distance from thetip of the needle to the surface of the collector from 12-15 cm,temperature in the range from 30° to 50° C., and flow rate in the rangefrom 0.4 mL/h to 2 mL/h to determine the conditions that would result information of the fibrous mats. Electric voltage was set to 23 kV butpreliminary results showed that fibers were also formed under otherconditions (e.g., at approximately 10 kV at flowrates in the range from0.4 to 20 mL/hr). The caseinate solutions were cloudy, an indicationthat all of the protein was not dissolved in solution, and viscous atroom temperature but became clear at 50° C. After experiments, the foilwrap holding the dry fibrous mat was removed from the rotating cylinderand placed in a desiccator to protect from moisture until furtheranalysis. The mats were typically 10 cm wide×4 cm long or were oval inshape with the longest diameter of 3 cm. The experiments were performedthree times for each set of conditions.

In a single experiment to create a polysaccharide-free caseinate fiber,the fibrous mat created from a 15% PUL and a 20% NaCAS aqueous solution(1:4) (NaCAS content of 84.2%) prepared as described above was soakedwith gradient ethanol/glutaraldehyde, starting with 95:5 (v/v) todehydrate the mat, remove the PUL and crosslink the remaining fiber.

Electron Microscopy: The fibrous mats were coated with a thin film ofgold and then examined using scanning electron microscopy (FEI,Hillsboro, Oreg.). The high-vacuum secondary electron imaging modes atan accelerating voltage of 10 kV and working distance of 12.5 mm wereused. The distribution of the fiber sizes for 100 fibers of the samplewere measured using the XT Docu preloaded software (Soft Imaging SystemGMBH).

Results and discussion. Electrospinning of PUL: Aqueous solutions of PULwere electrospun at concentrations of 5, 10 and 15% (w/w). Scanningelectron microscopy (SEM) of the electrospun PUL fibers are shown inFIG. 1. Electrospinning of the 5% solution did not result in fiberformation. Electrospinning of the 10% PUL solution resulted in fiberscontaining beads and donut shapes because of electro spraying.Electrospinning of the 15% PUL solutions surprisingly resulted in goodfiber formation.

A requirement for good fiber formation is entanglement of the polymerchains in solution. Stijnman et al. (2011) noted that polysaccharidesform fibers when the viscosity at 1000 s⁻¹ was in the range from about0.5 to about 6 PA s and the ratio of the concentration of apolysaccharide solution, c/ to the overlap concentration, c*, rangedfrom about 10 to 25; c*was calculated from the intrinsic viscosity andwas defined as that at which fibers begin to form; c/c* was reported as11.25 with c of 15 (w/w) % for PUL in agreement with this study(Stinjman et al. 2011; Kong, L., and G. R. Zeigler, Food Hydrocolloids,38: 220-226 (2013)).

The average diameters of the electro spun PUL fibers did not changesignificantly in size with an increase in concentration. Fiber diametersobtained from the 10% aqueous solutions at 50° C. were 211±63 nm(0.211±0.063 μm) in size. Fiber diameters obtained from the 15% solutionwere 192±46 nm (0.192±0.046 μm). Kong and Zeigler (2013) reported a mostprevalent fiber diameter range of 330-452 nm for electrospinning of PULfrom 12% (w/v) aqueous solution at 20° C. The sizes of the fibers dependon the viscosity of the solution, which may be manipulated bytemperature. In our study, experiments were conducted at 50° C. buthigher temperatures may result in fibers with even smaller diameters dueto the decrease in viscosity, facilitating spinning due to decreasedintermolecular interactions (Zhang et al. 2009). This is also balancedby increases in the conductivity and the surface tension of the solutionwith temperature which was not investigated here.

Electrospinning of caseinates with pullulan: It was not possible toelectro spin CaCAS or NaCAS in aqueous solution in the absence of aspinnable carrier. Electrospinning of aqueous solutions of 15% PUL with20% CaCAS in volume ratios of (1:1)(57% CaCAS) and (1:2)(72.7% CaCAS)was then conducted. All values of CAS in parentheses are on a dry basis.SEM of the electro spun fibers are shown in FIGS. 2A and 2B. Both SEMshowed relatively good fiber formation although the process may need tobe optimized through viscosity adjustment (T or c adjustments) toeliminate imperfections caused by electro spraying. The average diameterof the fibers for FIG. 2A is 263±52 nm (0.263±0.052 μm) and for FIG. 2Bis 159±42 nm (0.159±0.042 μm), indicating a decrease in fiber diameteras the amount of CaCAS was increased and the amount of PUL was decreasedin the solution.

Electrospinning of 30% PUL solutions with 20% CaCAS solutions in thevolume ratios of (1:1)(40CaCAS %) and (1:2)(57%) (FIGS. 2C and D) showeda size distribution of fibers and some electrospraying. FIG. 2E showsrelatively good fibers with surprisingly few imperfections for thevolume ratio of (1:4)(72.7%). The average fiber diameters for FIGS. 2C,2D, and 2E were 1020±627 nm, 387±110 nm, and 207±61 nm, respectively,showing a decrease in fiber diameter and the range in fiber diameter asthe amount of CaCAS in the solution was increased.

Successful electro spinning of CaCAS required that the concentration ofCaCAS exceed that of PUL to obtain fibers with the smallest diametersand with the least variation in size, surprisingly showing thatviscosity was the most effective variable for effecting changes in fibersize. The surface tension of CaCAS aqueous solution was increased uponaddition of PUL solution, which increased the surface tension necessaryto maintain the drop at the tip. Adjustments in surface tension candetermine the formation of fibers vs. beads but is also balanced byelectric field effects which affect the shape of the initial droplet. Inour study, the electric field was adjusted to a constant value of 23 kVbut adjustments in this variable would affect the balance with thesurface tension.

Aqueous solutions of 15% PUL were also electrospun with aqueoussolutions of 20% NaCAS in volume ratios of (2:1) (40% NaCAS), (1:1)(57%NaCAS), (1:2)(73% NaCAS), and (1:4)(84.2% NaCAS). The percentage ofNaCAS is also shown in parentheses. The corresponding SEM (FIG. 3A-D)shows the effects of adding progressively larger amounts of NaCAS to thesolution mixture. FIG. 3A shows relatively good fiber formation with noapparent imperfections. Increasing the amount of NaCAS resulted inincreasing amounts of defects and an apparent wider distribution offibers of various sizes and electro spraying. The average fiberdiameters for FIGS. 3A-3D are 308±56 nm, 319±29 nm, 341±149 nm, and465±207 nm.

The results for NaCAS indicated that electro spinning of the largeramounts of NaCAS required adjustments in temperature and solutionconcentrations to adjust viscosity to control fiber size and eliminateelectro spinning.

Under constant conditions of electric voltage and distance from the tipof the needle to the collector, the solution with the higherconductivity may result in a more elongated jet and fibers with smallerdiameter (Tan, S-H., et al., Polymer, 46: 6128-6134 (2005)). Both Ca andNa have approximately the same ionic radii, 0.99 A and 1.02 A,respectively, but it would be expected that Ca has twice the chargedensity of Na because of its ionic charge of 2. A comparison of FIGS. 2Band 3C, both with the same 1:1 ratio of PUL:CAS, showed that CaCASfibers had a smaller diameter than the NaCAS fibers. Surprisingly, thediameter of CaCAS fibers were 159±42 nm and that of the NaCAS fiberswere 341±149 nm. It was expected that NaCAS would have the smallerdiameter fibers. While Ca has twice the charge density of Na, CaCASsurprisingly had half the electrical conductivity of NaCAS (5396 μS forNaCAS in pure 20% solution vs. 2776 μS for CaCAS); without being boundby theory this is likely due to other various interactions whichaffected elongation forces on the jet under the electric field,resulting in a fibers with smaller diameter.

Freestanding casein fibers: FIG. 4 shows the NaCAS fiber reduced in PULby soaking with ethanol/glutaraldehyde. The fibers or fibrous mats ofPUL/NaCAS or CaCAS were soaked in gradient ethanol/H₂O containing 10%glutaraldehyde, the ethanol contents were decreased from 95% to 0% (v/v)in 4 hours, then the fibers and fibrous mats thus treated were washedwith DI water to remove free glutaraldehyde. The fiber, as shown earlierin FIG. 3D, had an average diameter of 487±192 nm. After theexperiments, removal of moisture and PUL are indicated on the fibers asa series of pores.

Conclusion: Our technology is the first that surprisingly createdelectro spun fibers from food proteins by using a food-gradepolysaccharide to facilitate molecular entanglement in solution andwhich required no treatments prior to electro spinning. Until now,spinnable polymers were used for protein electrospinning, or materialssuch as gelatin which are undesirable for food use. New types of foodsbased on dairy and other food proteins are envisioned which will allowinclusion of micronutrients, heat sensitive bioactives,probiotic/prebiotic blends into functional beverage and foodformulations, and possibly foods for medical use; foods to createsatiety, tailoring of the bioavailability of foods, and the developmentof edible sensors. The texture of food will also be affected as well asmodification of the water-binding properties of foods to help extendshelf-life. The removal of the carrier will also allow, which is notpossible with other technologies, exploration of free standing caseinatefibers, first for non food use and then for food use. This technologymay ultimately be found useful in the utilisation of surplus dairy andother ingredients to prevent waste.

All of the references cited herein, including U.S. patents, areincorporated by reference in their entirety. Also incorporated byreference in their entirety are the following references: Frinault, A.,et al., J. Food Sci., 62: 744-747 (1997); Konstance, R. P. et al.,Textural properties of casein(ate) gels and their utility in creatingsurimi-like seafood analogues, IN Chemistry of Novel Foods, ed. A. M.Spanier, M. Tamura, H. Okai, O. Mills, Allured Publishing Co., CarolStream, Ill., Chapter 16, pp 199-215, 1997; Southward,http://nzic.org.nz/ChemProcesses/dairy/3E.pdf; v/3E.pdf; U.S. Pat. No.8,066,932; U.S. Patent Application Publication 20060264140; U.S. PatentApplication Publication 20080110342.

Thus, in view of the above, there is described (in part) the following:

A method of forming a fiber mat, said method comprising (or consistingessentially of or consisting of) forming an aqueous solution of at leastone protein, at least one polysaccharide, and optionally a plasticizer,and electro spinning said aqueous solution onto a collector to form amat.

The above method, wherein said method utilizes a voltage sourceconnected to a needle, nozzle, tube, or pipe (with epical arrangements).

The above method, wherein the distance between the tip of said collectorand said needle, nozzle, tube, or pipe is about 12 cm to about 100 cm(e.g., 12 to 100 cm).

The above method, wherein said voltage source provides about 5 to about100 kV (e.g., 5 to 100 kV), preferably about 10 to about 30 kV (e.g., 10to 30 kV), more preferably 10 to about 23 kV (e.g., 10 to 23 kV).

The above method, further comprising (or consisting essentially of orconsisting of) soaking said mat in a solution containing at least onemember selected from the group consisting of ethanol, methanol, acetone,acetonenitrite, isopropyl alcohol, glutaraldehyde, formaldehyde, andmixtures thereof to remove said at least one polysaccharide.

The above method, further comprising soaking said mat in a solutioncontaining a chemical (e.g., an aldehyde, a mixture of an aldehyde likeformaldehyde and sodium cyanoborohydride) that fixes said at least oneprotein.

The above method, further comprising (or consisting essentially of orconsisting of) soaking said mat in a solution

containing a chemical (e.g., an epoxide, mixture of an epoxide andsodium hydroxide) that fixes said at least one polysaccharide.

The above method, further comprising (or consisting essentially of orconsisting of) soaking said mat in a solution containing a solvent(e.g., water at pH in range of 1-14) that dissolves said at least oneprotein.

The above method, further comprising (or consisting essentially of orconsisting of) soaking said mat in a solution containing a solvent(e.g., water at pH in range of 1-14) that dissolves said at least onepolysaccharide.

The above method, further comprising (or consisting essentially of orconsisting of) soaking said mat in a solution containing a mixture ofsolvents (e.g., mixture of water and an organic solvent, or two miscibleor immiscible organic solvents) that dissolves said at least oneprotein.

The above method, further comprising (or consisting essentially of orconsisting of) soaking said mat in a solution containing a mixture ofsolvents (e.g., mixture of water and an organic solvent, or two miscibleor immiscible organic solvents) that dissolves said at least onepolysaccharide.

A mat formed by the above method.

A mat formed by the above method, wherein said mat is suitable forfood-grade applications.

A mat formed by the above method, wherein said mat is not suitable forfood-grade applications.

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

We claim:
 1. A method of forming a fiber mat, said method comprising:forming an aqueous solution of at least one protein, at least onepolysaccharide, and optionally a plasticizer; and electrospinning saidaqueous solution onto a collector to form a mat, wherein the at leastone protein is a casein or caseinate protein.
 2. The method according toclaim 1, wherein said method utilizes a needle and a voltage sourceconnected to said needle.
 3. The method according to claim 1, whereinsaid method utilizes a voltage source connected to a needle, nozzle,tube, or pipe with epical arrangements.
 4. The method according to claim3, wherein the distance between the tip of said collector and saidneedle, nozzle, tube, or pipe is about 12 cm to about 100 cm.
 5. Themethod according to claim 2, wherein said voltage source provides about5 to about 100 kV.
 6. The method according to claim 2, wherein saidvoltage source provides about 10 to about 30 kV.
 7. The method accordingto claim 2, wherein said voltage source provides about 10 to about 23kV.
 8. The method according to claim 1, further comprising soaking saidmat in a solution containing at least one member selected from the groupconsisting of ethanol, methanol, acetone, acetonenitrite, isopropylalcohol, glutaraldehyde, formaldehyde, and mixtures thereof to removesaid at least one polysaccharide.
 9. The method according to claim 1,further comprising soaking said mat in a solution containing a chemicalthat fixes said at least one protein.
 10. The method according to claim1, further comprising soaking said mat in a solution containing achemical that fixes said at least one polysaccharide.
 11. The methodaccording to claim 1, further comprising soaking said mat in a solutioncontaining a solvent that dissolves said at least one protein.
 12. Themethod according to claim 1, further comprising soaking said mat in asolution containing a solvent that dissolves said at least onepolysaccharide.
 13. The method according to claim 1, further comprisingsoaking said mat in a solution containing a mixture of solvents thatdissolves said at least one protein.
 14. The method according to claim1, further comprising soaking said mat in a solution containing amixture of solvents that dissolves said at least one polysaccharide.